The proteins in breast milk may provide significant nutritional and other health benefits to infants and adults. Among the proteins that confer such benefits are .beta.-casein, which comprises 80% of the casein in human breast milk. The .beta.-casein in breast milk is believed to serve not only as a source of amino acids required for endogenous protein synthesis, but also as an anti-infective agent and an enhancer of calcium absorption. The addition of .beta.-casein as a supplement to infant formula or other nutritional products may, therefore, provide significant health benefits to infants and adults.
The commercial use of .beta.-casein as a supplement for infant formula or other nutritional products requires the production and isolation of large quantities of the protein at economically reasonable costs. Production of large quantities of .beta.-casein may be accomplished by recombinant production of the protein in bacteria, but such methods require that the protein be efficiently isolated from the complex mixture of other proteins and cell components present in the cell paste, homogenate or lysate obtained after fermentation. Current methods for isolating recombinant proteins often involve precipitating the protein from a suspension. Unless the precipitation is highly specific for the desired protein, however, such methods generally result in high levels of contamination with undesired proteins and other cell components originating from the host cells. In order to remove such contaminants, additional steps of separation and purification may be employed (e.g., HPLC, ion-exchange chromatography), but these are typically costly and time-consuming. In addition, some methods of protein isolation employ denaturing conditions and, therefore, these methods either result in poor yields of functional protein or require additional steps for renaturing the protein. See, e.g., U.S. Pat. No. 5,466,781.
Prior art methods for isolating .beta.-casein from genetically engineered bacterial cells typically involve precipitating the .beta.-casein from a supernatant derived from lysed or fractionated cells. For example, Simons, et al., Protein Eng. 6: 763-770 (1993), used genetically engineered E. coli to express bovine .beta.-casein. The protein, which accumulated in the periplasmic spaces of the bacteria, was released into a cell suspension by osmotic shock. After centrifugation of the suspension, the .beta.-casein in the pellet was resuspended in a cold water wash and centrifuged again. The .beta.-casein, present in the supernatant, was precipitated by acidification with acetic acid, filtered, and further purified by HPLC. Similarly, Hansson, et al., Protein Expression and Purif. 4: 373-381 (1993), used genetically engineered E. coli to express .beta.-casein. The .beta.-casein, present in a cell lysate, was precipitated with ammonium sulfate, dissolved in ethanolamine and 6M urea, and further purified by ion-exchange chromatography.
There remains, however, a need in the art for methods for isolating recombinantly produced .beta.-casein that are simpler and more effective than known techniques. The present invention addresses this need by providing methods for isolating large quantities of recombinantly produced .beta.-casein, that are considerably less costly and time-consuming, and result in higher yields with a high degree of purity.